Dioscin ameliorates inflammatory bowel disease by up-regulating miR-125a-5p to regulate macrophage polarization

Abstract

Purpose: Dioscin has been proven to have anti-cancer, anti-inflammatory, and anti-infection roles. However, the role of Dioscin in inflammatory bowel disease (IBD) and its related mechanisms is unclear and needs further study.

Methods: The colitis model in mice was established. After Dioscin (20, 40, or 80 mg/kg) treatment, the colon length was measured by a ruler. Histopathology, inflammatory cytokines, gut permeability, tight junction proteins, macrophage infiltration, macrophage polarization, and miR-125a-5p level were detected by hematoxylin-eosin staining, enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction (qRT-PCR), FITC-dextran, Western blot, and flow cytometry. In vitro experiments, after RAW264.7 cells induced by lipopolysaccharide (LPS)/interleukin-4 (IL-4), were treated with Dioscin and miR-125a-5p inhibitor, miR-125a-5p level, cell vitality, inflammatory cytokines, and M1/M2 marker genes were measured by qRT-PCR and MTT assay.

Results: Dioscin (20, 40, or 80 mg/kg) relieved DSS-triggered colitis and restrained the serum and colon of pro-inflammatory cytokines expression. Meanwhile, different concentrations' Dioscin weakened M1 macrophage polarization but facilitated tight junction protein expressions, M2 macrophage polarization, and miR-125a-5p level in colitic mice. Moreover, miR-125a-5p inhibitor reversed the modulation of Dioscin on miR-125a-5p expression, cell vitality, and inflammatory cytokines in lipopolysaccharide (LPS)-induced RAW264.7 cells. We further discovered that Dioscin restrained M1 marker gene (CD16) expression while intensifying M2 marker genes (CD206 and Arginase-1) expressions in vitro, which was reversed by miR-125a-5p inhibitor.

Conclusion: Dioscin modulated macrophage polarization by increasing miR-125a-5p, thereby improving the intestinal epithelial barrier function and reducing IBD.

Keywords: dextran sulfate sodium; dioscin; inflammatory bowel disease; lipopolysaccharide; miR-125a-5p.

FIGURE 1

Dioscin relieved DSS‐triggered colitis and restrained the serum and colon of pro‐inflammatory cytokines expression in a dose‐dependent manner. (A) The chemical structure of Dioscin. (B,C) Impact of Dioscin on the colon length of colitic mice was measured by ruler (n = 10 per group). (D) Impact of Dioscin on histopathology of distal colon from colitic mice was detected by hematoxylin–eosin staining (magnification ×100, scale bar = 200 μm). Infiltration of inflammatory cells and tissue injury (arrow and quadrangle). (E–G) Impact of Dioscin on pro‐inflammatory cytokines expression in the serum of colitic mice was detected by enzyme‐linked immunosorbent assay. (H) Impact of Dioscin on pro‐inflammatory cytokines expression in the colon tissues of colitic mice was detected by quantitative real‐time polymerase chain reaction (qRT‐PCR). Expression levels were normalized with glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). (I) Impact of Dioscin on gut permeability in the serum of colitic mice was detected by FITC‐dextran. The dose of Dioscin: 20, 40, or 80 mg/kg. All experiments have been performed in triplicate and data were expressed as mean ± standard deviation (SD). ^*** p < 0.001 vs. Control group; ^## p < 0.01, ^### p < 0.001 vs. dextran sulfate sodium (DSS) group. The figure represents at least 6 animal numbers/group

FIGURE 2

Different concentrations' Dioscin weakened M1 macrophage polarization but facilitated tight junction protein expressions and M2 macrophage polarization. (A,B) Impact of Dioscin on tight junction protein expressions in the colon tissues of colitic mice was detected by Western blot. Expression levels were normalized with GAPDH. (C,D) Impact of Dioscin on macrophage infiltration was detected by flow cytometry. (E‐H) Impact of Dioscin on macrophage polarization was detected by flow cytometry. (I) Impact of Dioscin on the miR‐125a‐5p level in the colon tissues of colitic mice was detected by qRT‐PCR assay. Expression levels were normalized with U6. The dose of Dioscin: 20, 40, or 80 mg/kg. All experiments have been performed in triplicate and data were expressed as mean ± standard deviation (SD). ^*** p < 0.001 vs. Control group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. DSS group. The figure represents 6 animal numbers/group

FIGURE 3

MiR‐125a‐5p inhibitor reversed the regulation of Dioscin on the miR‐125a‐5p level, cell vitality, inflammatory cytokines, and macrophage polarization in vitro. (A) Impacts of Dioscin and miR‐125a‐5p inhibitor (I) on the miR‐125a‐5p level in lipopolysaccharide (LPS)‐induced RAW264.7 cells were detected by qRT‐PCR assay. Expression levels were normalized with U6. (B) Impacts of Dioscin and miR‐125a‐5p inhibitor on the cell vitality in LPS‐induced RAW264.7 cells were detected by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. (C) Impacts of Dioscin and miR‐125a‐5p inhibitor on the inflammatory cytokines in LPS‐induced RAW264.7 cells were detected by qRT‐PCR assay. Expression levels were normalized with GAPDH. (D) Impacts of Dioscin and miR‐125a‐5p inhibitor on M1/M2 marker genes in LPS‐induced RAW264.7 cells were detected by qRT‐PCR assay. Expression levels were normalized with GAPDH. (E) Impacts of Dioscin and miR‐125a‐5p inhibitor on M2 marker genes in interleukin‐4 (IL‐4)‐induced RAW264.7 cells were detected by qRT‐PCR assay. Expression levels were normalized with GAPDH. Dioscin concentration: 150 ng/ml. All experiments have been performed in triplicate and data were expressed as mean ± standard deviation (SD). ^*** p < 0.001 vs. Control group; ^## p < 0.01, ^### p < 0.001 vs. LPS group; ^{^^} p < 0.01, ^{^^^} p < 0.001 vs. LPS + I group; ^†† p < 0.01, ^††† p < 0.001 vs. LPS + Dioscin + inhibitor control (IC) group; ^‡‡ p < 0.01, ^‡‡‡ p < 0.001 vs. IL‐4 group; ^ΔΔΔ p < 0.001 vs. IL‐4 + I group; ^§ p < 0.05, ^§§ p < 0.01 vs. IL‐4 + Dioscin + IC group. The figure represents three biological replicates with three technical replicates